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Spectroscopic Methods of

Analysis
UV-Vis spectroscopy
Electronic spectroscopy

UV_VIS

Principle

Electromagnetic
radiation

Sample

Quanlitative
Quantitative

(Absorption or emission)

UV_VIS

Electromagnetic radiation
Wave Properties
Particle Properties

Phng truyen

Direction of propagation

wavelength (cm, m, nm, A)


frequency (s 1)
the velocity of light c
c = . = 3 x 10 10 cm/s
wavenumber (cm 1)
= 1/ = /c

UV_VIS

The energy of a unit


of radition (photon)

E = h = hc/ = h.c.
E (eV, kcal/mol)
h: Planck constant = 6,626.10 34
J.s = 6,626.10 27 erg.s = 6,59 eV.s

Electromagnetic spectrum

UV-VIS

Electromagnetic spectrum
IR

UV-VIS

(Gamma rays)

Violet

X rays

Blue

380 nm

E = h = hc/ = hc

UV_VIS

Visible

Ultraviolet

Green

Yellow

Colorimetry

Infrared

Orange

NMR

Radio waves

Red
760 nm

NMR: Nuclear Magnetic Resonance


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Visible lights

UV-VIS

Colours of Visible Light

UV_VIS

i cng quang

Principle

Electromagnetic
radiation

Sample

Quanlitative
Quantitative

(Absorption or emission)

UV_VIS

Absorption and emission


(The excited state)

E1

Ee = E1 E0

(The ground state)

E0

E = Ee + Ev + Er
Ee : (electron energy)
Ev: (vibration energy)
Er: (rotation energy)

UV-Vis spectroscopy
Electronic spectroscopy
UV_VIS

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Instrument

UV_VIS

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Instrument

UV_VIS

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Instrument

UV_VIS

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Intrument

UV_VIS

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UV/VIS instrument

i cng quang

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Instrument

UV_VIS

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Instrument

UV_VIS

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Instrument

UV_VIS

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Grating
monochromator

polychromatic
radiation

monochromatic
radiation

UV_VIS

Typical grating monochromator

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Source

UV_VIS

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Detectors

UV_VIS

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VIS spectrum

UV_VIS

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IR spectrum

UV_VIS

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UV/Vis Spectra for Molecules and Ions

UV_VIS

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UV/Vis Spectra for Molecules and Ions


E

* Unoccupied level
Atommic Orbital
Occupied level
Molecular orbitals

* Energy
*

n - *

- *
-*

n-*

UV_VIS

Molecular orbitals

- * > n - * > - * > n - *


- * (150 nm)
n - * (150 200 nm)
- * (200 300 nm)
n - * (> 300 nm)
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Chromophores
Chromophores are groups of atoms within a molecule,
which absorb electromagnetic radiation.
The most important chromophores are:
Conjugated double bonds, such as:
O

Aromatic systems, such as:

UV_VIS

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Absorption of Simple Unconjugated


Chromophores
Chromophore

Notation of transition

max(nm)

C-C and C-H

~150

n *
n *

~185
~195
n * ~195
~300
~190

-bonded electrons
lone pair electrons
-O-N<
-S>C=O
>C=O

n *
n *

-bonded electrons
>C=C< (isolated)
*
>C=O
*
UV_VIS

of these,
mainly the
>C=O
absorption can
be seen in a
normal UV
spectrum

~190
~190
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Conjugated Systems Absorb at Longer


Wavelength
*
*

*
A

B
2

isolated
double bond

UV_VIS

LUMO - lowest unoccupied


molecular orbital
HOMO - highest occupied
molecular orbital

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two conjugated
double bonds

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Conjugated Systems Absorb at Longer


Wavelength
Increasing the conjugation shifts the absorption maximum
(lmax) towards longer wavelengths,
called a red or bathochromic shift. This has the advantage
that a standard UV detector is now able to observe this
absorption.
Decreasing conjugation has the opposite effect.. .a blue or
hypsochromic shift.
Also the intensity of the absorption (max) increases with
increasing conjugation.

UV_VIS

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Conjugated Systems Absorb at Longer


Wavelength

UV_VIS

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Absorption
Chromophore
Auxochrome
Bathochromic shift, red shift hypsochromic effect, blue
shift
Hyperchromic effect hypochromic effect

UV_VIS

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Solvent Choice
The choice of solvent in UV-Vis detection is
dependent on a few factors:
The solvent should not absorb light in the same
wavelength region as the substance that is being
analysed.
The solvent should be transparent at the wavelengths
that are being used in the analysis.
The solvent should not form a complex with the
analyte, subsequently disturbing the absorption
spectrum.
The solvent can be used to shift the absorption
wavelengths to either longer or shorter transition
wavelengths.
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Solvent Choice
Solvent

Lower wavelength limit (nm)

Water
Ethanol
Hexane
Cyclohexane
Methanol
Diethyl ether
Acetonitrile
Tetrahydrofuran
Dichloromethane
Chloroform
Carbon tetrachloride
Benzene

UV_VIS

205
210
210
210
210
210
210
235
245
265
280

solvents of choice
- no significant
interference

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Effect of Solvent
The solvent can influence the position (max) and
the molar absorptivity (max) of the absorbance
spectra, through changes in:
pH
Polarity
Electrolyte concentration

UV_VIS

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In Conclusion
Alkanes, alcohols and ethers cannot be observed
in UV-Vis, as the transitions involved are *
and n*
Ketones generally show weak n* transitions
and are visible in the UV region
Dienes and enons show strong * absorptions
and are also visible in the UV region.

UV_VIS

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Absorbance and Concentration: Beers Law


I

IO = IR + I A + I T = IA + I T
I

T (transmittance)
T = IT/Io or
T% = 100 x IT/Io

UV_VIS

A (Absorbance)
A = log I0/IT = log 1/T = log
100/ T% = 2 log T%

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Absorbance and Concentration: Beers Law


IR

A=bC
Io

IA

IT

The morlar absorptivity, (L x mol -1 x


cm -1)
A=abC
b: pathlength
C: concentration, mol/L

a The analytes absorptivity,


(L x g -1 x cm -1)
C: concentration, g/L or
mg/L

Depends on analyte, wavelength, temperature, matrix


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Example
A 5.00 104 M solution of an analyte is placed in a sample
cell that has a pathlength of 1.00 cm. When measured at a
wavelength of 490 nm, the absorbance of the solution is
found to be 0.338. What is the analytes molar
absorptivity at this wavelength?

UV_VIS

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Limitations to
Beers Law
Concentration
pH or dilution
Solvent
Temperature
Time
Ligand

UV_VIS

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Applications
Qualitative
Quantitative
One component
Multiple component

Determination of Equilibrium Constants


the acid-baz equilibrium constant

Stoichiometry of a Metal - Ligand Complex

UV_VIS

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VIS spectrum

UV-VIS

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UV-VIS

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UV-VIS

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Quantitative Analysis for a Single Analyte


(1) A = bc.
(2) Ac = bCc, Am = bCm ;
Am/ Ac = Cm/Cc Cm = Cc x Am/Ac
(3) Am = bCm, Am = b(Cm + Cc); Am- Am = bCc ;
Am/ Am - Am = Cm/C Cm = Cc x Am/ Am - Am
(4)
A(M1) A(M0)

C0

C1

C2

C3

A0

A1

A2

A3

C4
A4

C5
A5

M0

M1

A(M0)

A(M1)

A5
A4
A3
A2

Cm

A1

UV_VIS

C1

C2 C3 C4

C5

C, mol/L
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Example: Determination of Iron in


Water and Wastewater
The determination of Fe in an industrial waste stream
was carried out by the o-phenanthroline. Using the data
shown in the following table, determine the
concentration of Fe in the waste stream.

ppm Fe
0.00
1.00
2.00
3.00
4.00
unknown
UV_VIS

Absorbance
0.000
0.183
0.364
0.546
0.727
0.269

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Quantitative Analysis of Mixtures (Two


components)

UV_VIS

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Quantitative Analysis of Mixtures (Two


components)

A1 A1 A1 1 bC 1 bC
I

II

II

II

A2 AI2 AII2 I2 bC I II2 bC II


CI

A2 II1 A1 II2

I II I II
2

A2 1 A1 2
II

C
II

II

I II I II
2

UV_VIS

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Quantitative Analysis of Mixtures (Two


components) - Example

UV_VIS

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Quantitative Analysis of Mixtures (Two


components) Example

UV_VIS

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